Congenital cytomegalovirus (CMV) infection affects 1% of babies in the United States annually, causing mortality and permanent disabilities. Virus infection of the placenta precedes transmission to the fetus, and the routes of infection are linked to cytotrophoblast (CTB) interactions at the uterine-placental interface. Studies of biopsy specimens from early-gestation placentas revealed that CMV infects the uterus and spreads to differentiating/invading CTBs in anchoring villi and to progenitor cells in floating villi. Infected CTBs downregulate key differentiation molecules and impair invasiveness. Recent studies showed that IgG- virion complexes are transcytosed (mediated by the neonatal Fc receptor) across the syncytiotrophoblast and infect underlying CTBs. Immunostaining showed that sites of viral replication in the developing placenta correlate with expression of functional receptors upregulated as the cells differentiate. Surprisingly, CMV gB colocalized with caveolin-1 containing compartments in the syncytiotrophoblast, forming caveosomeswhere virions could accumulate at neutral pH. Caveolin-1-binding motifs in gB suggest virus-induced internalization. These novel observations establish that virions internalize in caveolae in naturally infected tissues and could explain why infection occurs throughout gestation. The long-term objectives are to understand mechanisms of transplacental transmission and prevent virus infection of the placenta and spread to the fetus. The hypothesis that gB promotes virion endocytosis in caveolae and virion transcytosis will be tested using specialized cells and chorionic villus explants and confirmed in naturally infected placentas. The specific aims are as follows. Aim 1. Examine dysregulated integrins and membrane-proximalfunctions[unreadable]cell adhesion, migration and invasion[unreadable]in differentiating CTBs infected with clinical CMV strains. Aim 2. Complete the analysis of molecules that function as CMV receptors in CTBs and specialized cells. Assess the role of caveolin-1-lipid rafts and gB in virion uptake. Correlate viral replication sites in infected placentas with developmental^ and spatially regulated receptors.Aim 3. Investigate virion transport in caveolar vesicles in polarized cells and evaluate cell-cell transmission of virions. Examine caveosomes isolated from naturally infected placentas. These studies will uncover mechanisms used by CMVto breach the placenta! barrier and reach the fetal compartment. Understanding these processes is the first step in the design of novel approaches to block viral spread and enhance the barrier function of the placenta to prevent damage caused by this important human pathogen in congenital disease.